Complex animals use hundreds of transcription factors (TFs) to accurately control cell-specific gene expression during the differentiation of specialized cell types within each organ. While genomic approaches have shown that many TFs bind thousands of overlapping regions, deciphering which DNA binding events and TF interactions are biologically meaningful remains a major challenge. The long-term goal of this application is to obtain a high-resolution understanding of the TFs, transcriptional mechanisms, and cis-regulatory logic used to ensure robust cell-specific EGF signaling during Drosophila development. Our experimental system is the transcriptional activation of the rhomboid (rho) protease that triggers EGF secretion from specific abdominal sensory organ precursor cells (SOPs) to induce metabolic cells (oenocytes) needed for animal growth and viability. Since only a subset of abdominal but not thoracic SOPs activate rho and the transcriptional levels of rho dictate the number of oenocytes specified, the regulation of rho serves as a great model to understand how regional- and tissue-restricted transcription factors are integrated to control robust cell-specific gene expression and phenotypic outcomes. Our findings during the first funding cycle of this grant revealed that: A) rho contains multiple cis-regulatory modules (CRMs) that activate abdominal SOP gene expression; B) A rho CRM contains numerous overlapping TF binding sites that directly integrate five TFs including an Abdominal-A (Abd-A) Hox complex containing the Extradenticle and Homothorax Hox cofactors and two neuronal transcription factors (Senseless and Pax2); C) AbdA-Senseless antagonism is a novel conserved Hox transcriptional mechanism that controls both EGF signaling in flies and blood cell proliferation and leukemia progression in mice. Building on these findings, this application has three aims: 1) Determine how the regional Abd-A Hox factor is integrated with the neural-restricted Pax2 factor to activate rho and assess which other Hox factors use Pax2 as a cofactor. 2) Define the role of additional neuronal transcriptional inputs that regulate rho in a specific subset of SOPs. 3) Use the underlying cis-regulatory logic to develop a bioinformatics algorithm to predict additional rho CRMs that ensure robust expression levels and phenotypes. Our approach combines the advantages of Drosophila genetics, non-biased mutagenesis reporter assays, and BAC genomic rescue assays with the speed of cell culture, biochemistry and bioinformatics. The successful completion of these aims has a high potential to uncover novel TF interactions that will open up new avenues of research. In addition, by coupling high-resolution mutagenesis studies with genomic rescue assays that provide a biologically meaningful readout, we will obtain new insights into how CRMs and transcription factors control robust cell-specific gene expression within a complex animal. Since the TFs and biological processes studied are highly conserved between flies and mammals, we are optimistic our mechanistic studies will continue to uncover new gene regulatory mechanisms relevant to human health and development.